JPH02295485A - Extraction of dna - Google Patents

Abstract

PURPOSE:To extract DNA from high-purity leukocyte with simple procedure in high efficiency by adsorbing and separating leukocyte from blood with a filter packed with fibrous substance having a specific diameter, washing the separated leukocyte to effect deproteinization, freezing the leukocyte together with the filter and thawing at room temperature. CONSTITUTION:DNA is extracted by conventional process from high-purity leukocyte produced by adding heparin and physiological salt solution to blood, passing the blood through a filter produced by filling a fibrous substance having an average diameter of <=10mum in a vessel to effect the adsorption of the leukocyte to the fibrous substance, washing the leukocyte adsorbed to the filter with a washing liquid composed of 10mM tris[hydroxymethyl]aminoethane and 1mM EDTA to remove proteins such as hemoglobin, freezing the leukocyte together with the filter at a low temperature (<=-20 deg.C) and thawing the frozen leukocyte at room temperature. The fibrous substance is preferably composed of polyester, polyamide, cotton, polyacrylonitrile, etc.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention is applicable to southern hybridization.
The present invention relates to an efficient DNA extraction method for extracting white blood cell DNA for use in, for example, ern hybridization. Background Art> In recent years, with the remarkable development of genetic manipulation technology, it has become possible to analyze various diseases at the genetic level. For example, genomic DNA is extracted from white blood cells in blood collected from peripheral blood vessels using a complicated method, and then analyzed using techniques such as Southern hybridization to diagnose genetic diseases. The Southern hybridization method uses cells (white blood cells, white blood cells,
After extracting DNA from lymphocytes, the DNA is cut using restriction enzymes, the cut DNA is electrophoresed, and then transferred to a membrane filter (referred to as plotting).
Then, after lightly washing the membrane filter and drying it in an oven, preferably performing an operation to reduce non-specific adsorption of the filter, hybridization is performed by adding 31P-labeled denatured probe DNA, and this is washed. Proviral DNA by autoradiography
It detects polymorphisms such as genetic defects, mutations, etc.
Traditionally, DNA has generally been extracted from leukocytes separated by the Ficoll method.

However, the above-mentioned Ficoll method requires that blood to which an anticoagulant such as heparin has been added is mixed with an equal amount of phosphate-buffered saline t & (
Add P B S, P H7.4) and mix well.
Gently inject this into the Spitz tube containing the separating agent,
The diluted blood sample was layered on top of the separating agent, and 400g30
After centrifugation, remove the upper layer without disturbing the intermediate layer containing white blood cells, collect the intermediate layer containing a small amount of the upper and lower layers, transfer it to another Spitz tube, add PBS, mix and wash. This time, centrifuge for 160glO, then remove the supernatant, add PBS, mix, and centrifuge for 100glO twice.Then, remove the supernatant and suspend in the required solvent.Finally, visualize! Calculate the number using a mirror and adjust the number of white blood cells to the required number. '', the operation was extremely complicated and required the operator's skill. Problems to be Solved by the Invention The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for extracting DNA that is easy to operate, does not require skill, and is efficient. .

<Means for Solving the Problems> In order to solve the above problems, the present invention (a) adsorbs and separates leukocytes from blood using a filter in which a container is filled with a fibrous substance having an average diameter of 10 lIm or less; do. (b) The white blood cells adsorbed on the filter are washed with a washing solution to remove proteins such as hemoglobin. (c) Freeze the leukocytes together with the filter at a low temperature of -20°C or lower, and then thaw at room temperature. We use a DNA extraction method that includes each step of the following steps. Effect> According to the present invention, when blood is passed through the filter, most of the white blood cells in the blood are adsorbed to the fibrous substance and separated from other blood components. Washing liquid such as TE (10 m
M tris[hydroxymethyl]aminoethane, 1mM
Washing through EDTA, pH 7.6) removes proteins such as hemoglobin. After freezing the white blood cells thus separated and washed together with the filter at, for example, -80°C,
Thaw the white blood cells by leaving at room temperature. The fibrous material was then treated with TE-+*ix (TE, 10mM Na c
L 1.5mM Mgclx, PH7.5)
When added, the white blood cells adsorbed to the fibrous material of the filter can be easily recovered from the fibrous material.

The procedure for extracting genomic DNA from collected white blood cells is as follows:
This can be done using a well-known method. Embodiments Next, embodiments of the present invention will be described.

The DNA extraction method of the present invention includes (a) an average diameter of 10 μm;
White blood cells are adsorbed and separated from blood using a filter whose container is filled with the following fibrous material. (b) The white blood cells adsorbed on the filter are washed with a washing solution to remove proteins such as hemoglobin. (c) Freeze the leukocytes together with the filter at a low temperature of -20°C or lower, and then thaw at room temperature. After thawing the white blood cells, the fibrous material is removed from the filter, a buffer such as TE-vix is added thereto, and the white blood cells are collected into a suitable test tube such as a Spitz tube. Furthermore, a surfactant such as 10% sodium dodecyl sulfate (SDS) and a proteinase are added to this.
K) and incubate at 65°C for 15 hours.
Add heat-treated RNA degrading enzyme (RNase A) to this and incubate at 37°C for 1 hour. This is then treated with a phenol reagent, and the DNA is purified by precipitation with ethanol. Adding proteolytic enzymes is
The reason for adding RNase A is to degrade cellular proteins that wrap DNA and proteins that connect DNA together (such as histones). for RNA
This is because if mixed with DNA, it may exhibit an inhibitory effect on probe DNA during hybridization.

Fibrous materials include polyester, polyamide, cotton,
Polyacrylic nitrile can be used, and polyester is particularly preferred since it has a high leukocyte recovery rate. The container filled with the fibrous substance has an opening through which the m-liquid can flow in and be discharged, and includes, for example, a syringe and a case of a commercially available leukocyte removal filter. A cleaning solution for cleaning white blood cells adsorbed on the filter is used to remove proteins such as hemoglobin attached to fibrous substances, and TE is used for -S. The freezing temperature of white blood cells is
Minus 2”C or less, preferably minus 20
Temperatures below ℃ are best, especially around minus 80℃. This is because rapid freezing at around -80°C will not damage the cells.

The reason why the white blood cells are left to thaw at room temperature is to make it easier for the white blood cells to peel off from the filter, and the buffer used after thawing is one that does not harm the cells and maintains a constant pH.
E-mjx is used. ED as a component of TE
TA is added to divalent cations by EDTA.
D, which easily binds to divalent cations by absorbing
This is to inhibit the reaction of NA degrading enzyme (DNAase). Ru. A surfactant is used to dissolve white blood cells, and when it is added, the white blood cells are quickly dissolved and internal DNA comes out, making the solution viscous. The surfactant may be non-ionic and biochemically used, and SD
In addition to S, NP-4 0 and Tween
etc. can also be used. A mixture of buffer and surfactant may be used to collect leukocytes, and a mixture of surfactant and protease may be used to lyse cells. Regarding the operations after collection of white blood cells after thawing, for example, please refer to the magazine "Clinical Examination J (VO1.32 no.4)
An appropriate method can be selected, such as the method introduced on page 393, left column, lines 4 to 37 of April 1988. 0,5I
10 d of physiological saline was added to this, and a peristaltic pump ( The white blood cells were adsorbed onto the polyester fibers by pouring the mixture while pulling it with the dial scale 3). Next, drain the blood in the syringe and remove the proteins such as hemoglobin attached to the polyester fibers.
It was removed by washing with TE10d (washing can be done with any amount of 4 to IOd). Next, the leukocytes were frozen together with the syringe at -80'C, and then allowed to stand at room temperature to thaw.

Then, the polyester fiber was taken out from the syringe, 4 ml of TE-mix was added thereto, and white blood cells were washed out and collected. The collected leukocytes were then treated with 10% SDS2.
00ul,'lli autolytic enzyme 50μg (5mg/d)
was added and incubated at 65°C for 15 hours. This was further heat-treated with RNA degrading enzyme 3d (1 0 *
/d), incubated at 37°C for 1 hour, treated with phenol reagent, white blood cells precipitated with ethanol, and DNA purified. The results of measuring the amount of white blood cells are shown. It can be seen that about 93% of leukocytes were adsorbed and separated.

Moreover, FIG. 1 shows the results of dissolving DNA in distilled water after ethanol precipitation and measuring the absorbance. DNA was recovered using Fichol High Bark (manufactured by Pharmacia) (Fic
It shows almost the same Bakun as when using the oll method),
It can be seen that DNA has been recovered. The extinction coefficient is IO
Although DNA appears at a wavelength of 260 nm, the amount of DNA recovered using this method is 33.5 ■/d, and when using the Picoll method (5.6 Ilg/More than one.

(Margins below) Table 1 [Example 2] In order to confirm that leukocytes recovered by this method can be used for Southern hybridization, DNA extracted using this method and Ficol method. was digested with the restriction enzyme By,9I, and hybridization was attempted using the gene encoding the β chain of thyroid stimulating hormone as a probe (approximately 500 bp). The resulting autoradio durums are shown in Figures 2 and 3. In both this method and the Ficol method, a single band was observed around 4.4 kb. This shows that DNA extracted using this method can be used for actual analysis in the same way as DNA extracted using the Ficol method. Note that the filter used in this method is the same as that in Example 1. In addition, the probe label is α(”P)-α
CTP was used and the dic translation method was used.

<Effects of the Invention> According to the present invention, the following effects can be achieved.

(1) Since leukocytes are adsorbed and separated onto a fibrous substance and then washed and recovered, the operation is simple and the purity of the leukocytes is high.

(2) Since the white blood cells are frozen and then thawed at room temperature,
The ability to adsorb fibrous substances is almost completely eliminated, making it possible to easily and efficiently recover almost the entire adsorbed white blood cells. (3) Since highly pure leukocytes can be easily and efficiently collected, DNA extraction can also be performed easily and efficiently.

[Brief explanation of drawings]

FIG. 1 is a graph showing the absorbance of DNA recovered by the method of the present invention and the Ficoll method, where 101 indicates the case when the present method was used, and 18- indicates the case when the Ficoll method was used. Moreover, FIG. 2 is an autoradiodrum showing the pattern of DNA digested by B, Q+, and FIG. 3 is a diagram for explaining D and Sn in FIG. Figure 1 Hll 2!11 126 24 @ 250
16＠ 276 2＠0 21＠ KoSO Hehi (n Taiyozuzusu D A: Ficoll method B: This method C: Marker procedure nefu seisho (method) (man 1 paper)

Claims (1)

[Claims] 1) (a) White blood cells are adsorbed and separated from blood using a filter in which a container is filled with a fibrous substance having an average diameter of 10 μm or less. (b) The white blood cells adsorbed on the filter are washed with a washing solution to remove proteins such as hemoglobin. (c) The leukocytes are frozen together with the filter at a low temperature of -20°C or lower, and then thawed at room temperature. A DNA extraction method comprising the steps of: 2) The DNA according to claim 1, wherein the fibrous material is made of polyester, polyamide, cotton, or polyacrylonitrile.
extraction method. 3) The method for extracting DNA according to claim 2, wherein the washing liquid is TE.